Abstract A prerequisite to the development of novel and powerful regenerative therapeutics is to reveal the mechanisms controlling the proliferation and differentiation of stem/progenitor cells. Stem cells are capable of proliferating indefinitely (self-renewal) and differentiating into one or multiple cell types (pluripotentiality). Progenitors are early descendants of stem cells that have pluripotentiality, but cannot divide indefinitely. Stem/progenitor cells are critical for homeostatic tissue maintenance and repair. However, the identity, origin, and role in renal regeneration of kidney stem/progenitor cells remain controversial. In vivo lineage tracing is a powerful technique to discover stem/progenitor cells in their native context. With this technique, a few mouse kidney stem/progenitor cell markers have been identified, including Six2, Lgr5, and Pax8. The stem/progenitor cells expressing these markers differentiate into various cell types, but not the collecting duct cells. Therefore, the stem/progenitor cells of the collecting duct remain mysterious, because a specific lineage-tracing marker is still not available. The collecting duct system is the final part of the kidney to influence the body's electrolyte, acid- base, and fluid balance. It has structurally and functionally distinct principal cells (PC), ?-intercalated cells (?- IC), and ?-intercalated cells (?-IC). In vitro studies suggest that ?-IC are putative stem cells and give rise to ?- IC and PC, while PC are terminally differentiated. However, the PI's recent in vivo studies with collecting-duct- specific histone H3 K79 methyltransferase Dot1l knockout mice (Dot1lAC) overturned this traditional view. Without Dot1l function, the cells expressing Aqp2, a well established PC marker, give rise to both ?-, and ?-IC. In this proposal, the PI proposes to extend and solidify these novel findings. In particular, the PI intends to discover derivation of IC from Aqp2-expressing cells occurs naturally (i.e, without need of Dot1l deletion). This would lead to identification of Aqp2 as the missing progenitor marker of collecting duct cells (Aim 1). The PI also intends to define Dot1l as a critical novel epigenetic regulator of collecting duct differentiation (Aim 2). Finally, the PI proposes to discover how Dot1l plays its regulatory role. In this regard, he will unearth HDAC2 as a new partner and negative regulator of Dot1l. Dot1l and HDAC2 mutually inhibit their opponent's function by restricting association with DNA. The PI will directly test the hypothesis whether HDAC2 deletion rescues the Dot1lAC phenotype (Aim 3). All of the required key reagents including multiple published and unpublished double and triple transgenic mouse models have been exclusively developed in the PI's lab for this project. Cellular, molecular, genetic, reno-physiological, electro-physiological, pathological, and electron microscopic approaches will be used. This proposal truly has high significance, impact and novelty because if successful, it will establish Aqp2 as a novel progenitor cell marker specific for renal collecting duct, Dot1l as the first epigenetic player in PC and IC differentiation, and HDAC2 as a novel partner and regulator of Dot1l.